Determination of red blood cell parameters Flashcards
Outline the procedure for venepuncture
apply a tourniquet and select a vein, then clean the area above it using an alcohol swab; insert the butterfly needle at 45deg, waiting for flashback; when you get flashback, insert the tube to the holder, and allow it to fill; when full, remove the tube and the tourniquet and then remove the butterfly needle
Describe the sample preparation
Before each of the three analysis tasks in parts 2, 3 and 4, you will need to prepare a small amount of blood on a piece of parafilm, as it isn’t easy to access the blood inside your EDTA tube.
This can be done by using a Pasteur pipette by drawing up a small amount and dropping 7-10 drops on the parafilm.
It is important to do this before each test! As time goes on the red cells sink to the bottom of the sample and you won’t be analysing mixed blood.
Describe how you perform the RBC count
dilute the blood 200-fold using a 5uL capillary pipette to mix 5uL of blood with 995uL isotonic saline; place a cover slip on the improved haematocytometer slide, leaving a small gap exposed at the top; in the gap place a few drops of the diluted blood so that it fills the space between the slides; examine the slide under a microscope and using edge convention, count the number of cells in 5 non-adjacent medium squares
Describe how you obtain a value for Haematocrit experimentally
fill a capillary tube with blood from a fresh sample on parafilm, before sealing the end with wax, before centrifuging for five minutes; use the haematocrit reader to give the percentage haematocrit
Describe the haematocytometer slide
Before starting examine the “Improved Neubauer” haemocytometer slide you will be using. The central portion is depressed so that it will lie just 0.1 mm below the glass cover, and it incorporates a calibrated ruled area.
The rulings define a central square with 1 mm sides, divided into 25 smaller squares (0.2 mm sides) each further divided into 16 squares with 0.05 mm sides.
Taking into account the 0.1 mm thickness, the volume above the large square is 0.1 mm3, a medium square 0.004 mm3, and one of the small squares 0.00025 mm3.
What is the volume of the 5 squares
Typically we sample 5 medium squares (80 small ones) spread around the slide, giving a total volume of 0.02 mm3. However the cell density in normal blood would be too great to count accurately so it is diluted 200-fold first, meaning this volume represents 0.0001 mm3 of undiluted blood. The number of red cells counted over 80 small cells is thus multiplied by 104 to reach the number of cells per mm3 in the blood sample, or by 1010 for the number per litre
Describe edge convention
One problem is what to do about cells touching the edge of a square – should they be counted as in that square or the adjacent one? To resolve this we use an “edge convention” such that any cell touching the bottom or right sides are counted in and those touching the other two sides (hatched cells in figure below) are not counted.
How is RBC count calculated
The red cells on 80 small squares are counted. If the number of cells counted is N, then the red cell count (RBC) will be N × 1010/litre of blood.
For example, if N = 506, the RBC would be 506 × 1010/L. This is more usually expressed as 5.06 × 1012/L.
What are the normal ranges of RBC count for males and females
Normal Range (Mean) of RBC count (/L):
Male 4.3 – 5.9 (5.1) × 1012
Female 3.7 – 5.3 (4.5) × 1012
What is meant by Haematocrit and how is it determines
Haematocrit (Hct) is the percentage of the volume of a sample of blood occupied by the red cells. Shake the blood sample. Transfer by capillary attraction enough blood to the centrifuge capillary tube to fill it to about 2 cm from the other end. Now seal this end using the special wax provided. Place the tube in one of the grooves of the centrifuge with the sealed end outwards and note the number of the groove that your sample is in. Do not start the centrifuge, it will be operated for you to give 12,000 times the force of gravity for 5 minutes.
After spinning, the blood will have separated into a layer of tightly packed red and white cells at the bottom and plasma at the top. As the tube is of uniform bore, the ratio of the length of the red cell column to the total length of the cells and the plasma is the haematocrit.
What are the normal ranges for Hct in males and females
Normal Range (Mean) Hct (%):
Male 40-52 (46)
Female 35-47 (41
How do we determine Hb concentration experimentally
Haemoglobin concentration in the anticoagulated blood sample is measured using the HemoCue Hb 201+ system, illustrated above.
What are the normal ranges for Hb concentration in males and females
Normal Range (Mean) (g/L):
Male 133 – 177 (155) g/L
Female 117 – 157 (137) g/L
How is MCV calculated
Hct/
(100xRBC)
fL
How is MCHC calculated
(100xHb)/
Hct
g/L
